The goal of this project is to characterize the mechanisms governing the evolution of Vibrio cholerae strains with epidemic potential within the context of a natural endemic environment. Emphasis will be placed on molecular genetic analysis of the role of vibriophages (bacterial viruses that grow on Vibrio species) in horizontal transfer of virulence-related genes, and in modulating V. cholerae population dynamics in the environment. Vibriophages will be isolated from 10 environmental surveillance sites in Bangladesh and characterized with respect to their host range, environmental prevalence, and ability to package and transfer V. cholerae DNA encoding virulence-related genes. Any phage that correlates negatively or positively with the presence of specific subgroups of V. cholerae will be monitored more closely with specific PCR or nucleic acid probes. In this way we hope to assess the phages ability to modulate the prevalence of specific V. cholerae strains, thereby affecting the ability of these strains to cause outbreaks of cholera. Environmental and clinical V. cholerae strains will also be collected, analyzed by microarray, DNA sequencing, and genetic fingerprinting methods to establish the phylogenetic relationships among the strains. The virulence gene content of the collected strains will be determined, and variant alleles of certain genes will be tested for their ability to mediate virulence-related functions (e.g., intestinal colonization). Clinical isolates that do not possess known colonization factor genes will be subjected to genetic analysis in order to identify the novel determinants carried by these strains. V. cholerae present in cholera stools have been reported to show increased infectivity in infant mice. We will examine this phenomenon using a variety of approaches including physiology assays, expression profiling with V. cholerae microarrays and quantitative chemotaxis assays. The results of this project will not only provide a full understanding of the processes by which pathogenic V. cholerae strains emerge, evolve, and transmit, but will also guide the development of surveillance and preventive measures that can be used to combat cholera in a cholera endemic area.